Initialization

Importing Data. Selecting needed samples for the run.

# Reading the full dataset
GeneExpression <- readRDS("GE.Rdata")
GMTFileName <- "c5.go.bp.v7.5.1.symbols.gmt"

# selecting the samples we need
#GeneExpression <- GeneExpression[GeneExpression$samples$cell_type == "cd117",]
GeneExpression <- GeneExpression[GeneExpression$samples$lib.size > 100000,]

# setting up main parameters
FactorsList <- c("treatment")

# setting up parameters for GO analyses
CurrentFactor <- 1
FDRLimit <- 0.1
GeneSetOfInterest <- "GOBP_CEREBELLAR_CORTEX_FORMATION"

Normalization to middle line.

Unsupervised PCA

## Warning in brewer.pal(nlevels(col.group), "Set1"): minimal value for n is 3, returning requested palette with 3 different levels

Model for DEG calculations

# Start of the model

ModelDesign <- model.matrix(~treatment, data = GeneExpression$samples)
ExpressionValues <- voom(GeneExpression, ModelDesign, plot = T)

ModelFit <- lmFit(ExpressionValues, ModelDesign) %>% eBayes
Result <- topTable(ModelFit, number = Inf, sort.by = "logFC")
## Removing intercept from test coefficients
#End of the model

DEG calculations results

##      (Intercept) treatmentRBD
## RBD1           1            1
## RBD2           1            1
## RBD3           1            1
## WT1            1            0
## WT2            1            0
## WT3            1            0
## attr(,"assign")
## [1] 0 1
## attr(,"contrasts")
## attr(,"contrasts")$treatment
## [1] "contr.treatment"
##        (Intercept) treatmentRBD
## Down            12           24
## NotSig        1464        19048
## Up           17675           79

## Warning in instance$preRenderHook(instance): It seems your data is too big
## for client-side DataTables. You may consider server-side processing: https://
## rstudio.github.io/DT/server.html

Supervised PCA

## Warning in brewer.pal(nlevels(col.group), "Set1"): minimal value for n is 3, returning requested palette with 3 different levels

##     Standard deviation Proportion of Variance Cumulative Proportion
## PC1           26.78295                0.29917               0.29917
## PC2           22.51462                0.21141               0.51057
## PC3           20.89927                0.18216               0.69274
## PC4           19.91570                0.16542               0.85816
## PC5           18.44207                0.14184               1.00000

GO

## Warning: One or more parsing issues, see `problems()` for details
## Rows: 7658 Columns: 25
## -- Column specification --------------------------------------------------------
## Delimiter: "\t"
## chr (25): X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, ...
## 
## i Use `spec()` to retrieve the full column specification for this data.
## i Specify the column types or set `show_col_types = FALSE` to quiet this message.
##   Negative Positive
## 1        0        1
## 2        0        1
## 3        0        1
## 4        1        0
## 5        1        0
## 6        1        0
## attr(,"assign")
## [1] 1 1
## attr(,"contrasts")
## attr(,"contrasts")$GroupFactor
## [1] "contr.treatment"
##           Contrasts
## Levels     TreatmentEffect
##   Negative              -1
##   Positive               1
## [1] "GO pathways analyzed: 7649"
## [1] "Affected GO pathways (Up + Down, FDR < 0.1 ): 0 + 0"
## Joining, by = "GO.ID"

Selected GO

Execution start time: 2022-02-14 12:41:49
Execution end time: 2022-02-14 12:42:35